Figure 8.

CaMKII-binding to the Ca_V1.3 NTD is required for high K⁺-induced CREB Ser¹³³ phosphorylation. A, CaMKIIα/β knockdown or re-expression/rescue has no effect on high K⁺-induced increases of somatic Ca²⁺. Cultured hippocampal neurons were transfected with mApple only (n = 17), mApple with CaMKIIα/β shRNA (n = 23), mApple/CaMKIIα/β shRNA with shRNA-resistant CaMKIIα^R-WT (n = 26), or CaMKIIα^R-V102E cDNA (n = 17), respectively. Transfected neurons were then monitored for Ca²⁺ influx in response to 40 mm K⁺-induced depolarization (black arrow; see Fig. 7A). B, the CaMKIIα-V102E mutant does not support nuclear signaling. Expression of CaMKIIα/CaMKIIβ shRNAs significantly reduces nuclear CREB phosphorylation following 40 mm KCl treatment. This reduction in CREB phosphorylation is rescued by co-expression of shRNA-resistant CaMKIIα^R-WT, but not CaMKIIα^R-K42R (kinase-dead) or CaMKIIα^R-V102E (deficient in Ca_V1.3 NTD binding). Each data point represents analysis of a single cell collected from 3–4 independent neuronal cultures/transfections. Pooled data were analyzed by one-way ANOVA followed by Tukey's multiple-comparison test. ***, p < 0.001; ns, not significant (p > 0.05). All confocal images show a 40 × 40-μm area. Error bars, S.E.